1. Field of the Invention
The present invention relates generally to a composition which is therapeutic and/or cosmetic to the tissue to which it is applied. The composition of the present invention preferably modifies or appears to modify the physical characteristics of the tissue to which it is applied, and the tissue being modified is preferably mammalian skin tissue. The present invention relates generally to the use of 7-hydroxylated steroids in such compositions useful in prevention and/or to treating the effects of aging and of the action of ultra-violet radiation (UV) on the skin. In an alternative embodiment of the present invention, the invention may include peptides which are formulated at therapeutically effective concentrations to increase the appearance skin. The peptides are preferably soluble in an aqueous solution, and more preferably are comprised of small peptides (usually less than about 10 amino acids in length). It has been found that the most preferred peptides are ones which correspond to or are homologous with portions of elastin, particularly with peptides which correspond to or are homologous with portions of elastin endogenous to the tissue being treated. Accordingly, it has been found that digestion of elastin, for example hydrolytic or site specific enzymatic cleavage of elastin results in peptides which are particularly suitable for use in the present invention. Accordingly, the peptides which result from digesting can be used directly in the therapeutic formulation of the present invention or may be analyzed and sequenced and synthesized by those methods known in the art (i.e. solid state, liquid, and over expression). As used herein, the term xe2x80x9cpeptidexe2x80x9d is not meant to convey any meaning regarding the precursor material or methods utilized to synthesize or make the peptides. Additionally; the term xe2x80x9celastin peptide fragmentxe2x80x9d in either singular or plural form refers to the fact that the peptide or amino acid sequence being discussed corresponds to, is the biological equivalent of, or is homologous with, a portion of elastin, more specifically to a portion or fragment of elastin endogenous to the animal being treated. However, the term xe2x80x9celastin peptide fragmentxe2x80x9d is not meant to convey any meaning regarding the source or starting material or method of use to make the elastin peptide fragment. As stated above, peptides of the present invention are preferably formed by enzymatic cleavage of elastin and are even more preferably formed by cleavage of elastin with thermolysin to form hydrophilic elastin derived peptides. It is also preferable that the peptides of the present invention are at a therapeutically effective concentration within the therapeutic or cosmetic composition, wherein the therapeutically effective concentration is in a range of about 0.0002% to about 90% by weight of peptide, more preferably in a range of about 0.05% to about 50% peptide, even more preferably in a range of about 0.5% to about 10% peptide, even more preferably about 1.5% peptide, and most preferably about 1.3% hydrolyzed elastin peptide. The therapeutic composition of the present invention can be formulated as a cosmetic preparation to be applied topically to a patient""s skin, such as in an emulsion, lotion, spray, powder, ointment, cream, or foam or in other suitable pharmaceutical vehicles or carriers commonly known in the art for other types of administration (i.e., subcutaneous). The delivery system of the present invention is preferably a topical delivery system but also may be a subcutaneous, transcutaneous, oral, nasal, aerosol, or patch delivery system.
2. Background and Description of the Related Art
Skin, in particular mammalian skin consists of a number of overlapping layers of cells. The outermost layer of mammalian skin is called the stratum corneum. This layer protects mammalian skin from physical and atmospheric harm, acting as a barrier to external dangers. The degree of softness or texture of the stratum corneum is directly dependent on its moisture content. However, it has been found that, in the lower layers of the skin, degenerative changes which occur with age are not caused principally by a lack of moisture. Therefore, even though the texture and appearance of the skin is dependent on the moisture content of the skin, other factors have been shown to influence the overall appearance and texture of the skin. For example, it has been found that the loss of elasticity in the skin decreases the tone and turgor of the skin. It is speculated that the decrease in skin tone and turgor occurs through degradation of certain complex polypeptides which are present in the skin. These complex polypeptides include elastin and collagen, among others.
Elastin is a highly cross-linked complex polypeptide and is a major component of elastic fibers present in the skin and connective tissue of animals. Elastin appears to be primarily responsible for the physiological elasticity of tissue. In normal mammalian skin, specifically human skin, elastic tissue proteins represent a relatively small fraction of the total dermal proteins, but play a very important role in maintaining or improving the skin tone and structure. Elastin itself is the main protein substance present in elastic fibers and occurs in tendons, blood vessels, and connective tissue. When isolated from these sources, it is normally in the form of a brittle, fibrous, yellowish material which is insoluble in water, alcohol and ether but is somewhat soluble in concentrated aqueous alkali metal hydroxide solutions. The dense cross-linked structure of elastin makes it very difficult to solubilize. There have been many attempts to solubilize elastin and create cosmetic agents from the solubilized elastin. To date, these techniques have not found a large degree of commercial success. Attempts to solubilize are described for example in a U.S. Pat. No. 4,327,078. However, it has been found that elastin is only slightly absorbed by the skin and does not sufficiently penetrate the skin to produce substantial benefits to the skin.
The formation of steroidal hormones, their interrelations and their functions have been widely described. The functions of pregnenolone (PREG) and of dehydroepiandrosterone (DHEA) as well as certain of their derivatives have been described in the International Published Application No. WO 94/08588, the teachings of which are hereby incorporated herein by reference thereto in its entirety.
Dihydroepiandrosterone and its sulphate derivative (S-DHEA) circulate in large quantities in humans, but its level reduces with age. It has thus been proposed to use S-DHEA in a cosmetic composition for topical application intended to treat certain signs of aging. The many effects of DHEA have been described, and certain of them oppose the processes and pathologies associated with aging. Despite numerous experiments, no completely satisfactory explanations for the effects of DHEA have been put forth, and the therapeutic use of DHEA has revealed undesirable secondary effects, particularly for women, as a potential precursor of androgynous hormones.
It has been shown that the 7-hydroxylated derivatives of PREG and of DHEA are formed by an enzyme system present in many tissues and organs, including the skin where they encourage mechanisms associated with immunity. Just like the amounts of DHEA circulating, the activity of these hydroxylating enzymes reduces with age.
Recent work relating to the cutaneous modifications caused by age or by UV and their medical treatment specifically considers retinoic acid, the xcex1-hydroxy acids and DHEA, but does not consider 7-hydroxysteroids.
The production of 7-hydroxylated derivatives of DHEA has been known for a long time in the tissues of the human fetus, in the amniotic epithelium, the human liver, the testicles and human epididymus and in the human pre-adipocytes. For the purposes of the present invention, it is preferable that the 7xcex1-hydroxylated derivatives of DHEA be isolated by the above-identified sources, even more preferable that the derivatives of DHEA be selectively 7-hydroxylated (7xcex1-hydroxylated) by Fusarium moniliforme. Furthermore, the amounts of 7xcex1-hydroxy-DHEA in circulation in pre-menopausal women have been measured at 200-300 pg/ml, and 3xcex2, 7xcex1 dihydroxy-5xcex1-androstan-17-one (7xcex1-hydroxy-isoandrosterone) has been identified in human urine. More recently, the phenomenon of 7-hydroxylation has been extended to other steroids that have, as does DHEA, a 3xcex2-hydroxylated structure (e.g. PREG, 5xcex1-androstane-3xcex2, 17xcex2-diol, 3xcex2-hydroxy-5xcex1-androstan-17-one and 3xcex2-hydroxy-5xcex1-pregnan-20-one).
Work on 7-hydroxylated steroids has proved that they are devoid of the distinctive hormonal effects of either androgens or estrogens or of effects on the secretion of hypophysial hormones. These results taken together have lead to the 7-hydroxylation of steroids being considered as a terminal hormonal deactivation process that leads to the urinary and biliary excretion of the 7-hydroxylated steroid products. It is only very recently that it has been possible to explain, in part, the many effects observed with DHEA by the immunostimulatory properties of its 7-hydroxylated derivatives. The antiglucocorticoidal properties shown by the 7xcex1- and the 7xcex2-hydroxy-DHEA have been proven and extended to other 7-hydroxylated steroids, such as those described in the International Publications Nos. WO 93/20687 and WO 94/08588, the teachings of which are herein incorporated by reference as if set forth in their entireties herein, for their role in triggering immune processes.
It appears that DHEA and the production of its 7-hydroxylated derivatives reduce with age. During aging, the supply of hormonal steroids to the skin is therefore modified with a predominance of glucocorticoids the effects of which as promoters of cutaneous aging are known.
Consequently, a localized supply of 7-hydroxylated steroids endowed with a particular antiglucocorticoid effect and yet natural allows the treated skin to be restored within its young age steroidal context.
The effects of 7-hydroxylated steroids and their derivatives on the cells that constitute the human skin and which are affected during aging or after UV irradiation were studied. Evidence was found that the effects of glucocorticoids leading to cellular apoptosis are obstructed by 7-hydroxylated steroids and that their action on the cutaneous cells is translated into beneficial and protective effects.
In a surprising way, the results obtained with the compounds of the invention do not correspond to those classically expected with steroidal hormones. In effect, the process of hydroxylation carried out by the body on PREG and DHEA is irreversible and due to this fact, the classic steroidal hormones can no longer be produced from the 7-hydroxylated derivatives.
As a consequence, the use of 7-hydroxysteroids for cosmetic purposes to treat or to prevent the cutaneous effects of aging has remarkable advantages compared with previously known steroids of the previously known cosmetic compositions.
The present invention is further directed to a composition for improving tissue texture, wherein the composition includes an elastin peptide which is synthesized by selectively cleaving elastin. Preferably the composition includes a pharmaceutical delivery system and the elastin is derived from animal tissue. More preferably, ligamentum nuchea is the source of the elastin starting material used in the present invention. The elastin of the present invention is preferably selectively cleaved by enzymatic digestion of the elastin with thermolysin.
The present invention is further directed to a method of enhancing the functionality, tone, turgor, and elasticity of the tissue to which it is administered. With particularity to skin tissue, the appearance of the skin is enhanced as a consequence of improving the elasticity of the tissue to which the composition is applied. In one embodiment it is most preferable that the method include the step of stimulating endogenous production of elastin in the patient to which an elastin fragment peptide is being applied. It is preferable that the administration step be comprised of a number of separate administration steps which are repeated most preferably twice daily over a predetermined time, wherein the predetermined time exceeds one week of daily administration of the composition, more preferably two weeks, and most preferably at least a month of daily topical application (with twice daily of the composition administration over the month being even more preferable.). As with the composition of the present invention, in the method of the present invention, it is preferable that the elastin peptide fragment be comprised of peptides having a molecular weight of less than about 10,000 Da, more preferably 90% of the peptides have a molecular weight of less than about 10,000 Da. It is even more preferable that the elastin peptide fragment be comprised of peptides having a molecular weight of less than about 3,000 Da and most preferably less than about 1,000 Da. In fact, it has been found that the preferred molecular weight range of peptides utilized in the present invention is in the range of about 100-1,000 Da; more preferred about 150-800 Da; even more preferred about 180-600 Da; and most preferably the therapeutic or cosmetic composition includes peptides having a molecular weight in the range of about 188-585 Da.
It has also been found that the peptides which best accomplish an increase in tissue elasticity and turgor are ones which correspond to or are homologous with portions of elastin, particularly with peptides which correspond to or are homologous with portions of elastin endogenous to the tissue being treated. Accordingly, it has been found that digestion of elastin, for example hydrolytic or site specific enzymatic cleavage of the elastin results in peptides which are particularly suitable for use in the present invention. Accordingly, the peptides which result from digesting can be used directly in the therapeutic formulation of the present invention or may be analyzed and sequenced and synthesized by those methods known in the art (i.e. solid state, liquid, and over expression). As used herein, the term xe2x80x9cpeptidexe2x80x9d is not meant to convey any meaning regarding the precursor material or methods utilized to synthesize or make the peptides. Additionally, the term xe2x80x9celastin peptide fragmentxe2x80x9d in either singular or plural form refers to the fact that the peptide or amino acid sequence being discussed corresponds to, is the biological equivalent of, or is homologous with, a portion of elastin, more specifically to a portion or fragment of elastin endogenous to the animal being treated. However, the term xe2x80x9celastin peptide fragmentxe2x80x9d is not meant to convey any meaning regarding the source or starting material or method of use to make the elastin peptide fragment. As stated above, peptides of the present invention are preferably formed by enzymatic cleavage of elastin and are even more preferably formed by cleavage of elastin with thermolysin to form hydrophilic elastin derived peptides. It is also preferable that the peptides of the present invention are at a therapeutically effective concentration within the therapeutic or cosmetic composition, wherein the therapeutically effective concentration is in a range of about 0.0002% to about 90% by weight of peptide, more preferably in a range of about 0.05% to about 50% peptide, even more preferably in a range of about 0.5% to about 10% peptide, even more preferably about 1.5% peptide, and most preferably about 1.3% hydrolyzed elastin peptide. The therapeutic composition of the present invention can be formulated as a cosmetic preparation to be applied topically to a patient""s skin, such as in an emulsion, lotion, spray, powder, ointment, cream, or foam or in other suitable pharmaceutical vehicles or carriers commonly known in the art for other types of administration (i.e., subcutaneous). The delivery system of the present invention is preferably a topical delivery system but also may be a subcutaneous, transcutaneous, oral, nasal, aerosol, or patch delivery system.
The present invention is further directed to a composition for improving tissue texture, wherein the composition is comprised of an elastin peptide which is synthesized by selectively cleaving elastin. Preferably the composition includes a pharmaceutical delivery system and the elastin is derived from animal tissue. More preferably, ligamentum nuchea is the source of the elastin starting material used in the present invention. The elastin of the present invention is preferably selectively cleaved by enzymatic digestion of the elastin with thermolysin. This thermolytic cleavage preferably results in a elastin peptide fragment or fragments having a molecular weight of less than about 10,000 Da, more preferably less than about 3,000 Da, even more preferably less than about 1,000 Da. A preferred composition of the present invention is one in which the elastin peptide fragment or fragments have a molecular weight of less than 1,000 Da. It is also preferable that the elastin peptides fragment or fragments include a sequence according to the formula of R1-Prolyl-Glycine-R2, wherein R1 is an amino acid or amino acid sequence (peptide) which is at the amino terminal and R2 is an amino acid or amino acid sequence (peptide) which is at the carboxyl terminal.